Experiment 2 Valinomycin 300 nM
Briefly, the gRNA pooled library in the vector lentiCRISPRv2 was amplified in Endura competent cells, and NGS sequencing performed to determine sgRNA distribution.Lentivirus was then generated in HEK293T cells and virus titer was determined. Target YFP-Parkin-MEFs were lentivirally transduced with the library at 0.3 MOI, treated or not with two concentrations of valinomycin (300 nM or 600 nM) for three rounds of treatment and enriched by clonogenic survival, for pools of cells expressing multiple gRNAs targeting the same gene. Genomic DNA was harvested and prepared for NGS.